Megabase-scale human genome rearrangement with programmable bridge recombinases.

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Bridge recombinases are RNA-guided DNA enzymes capable of programmable DNA manipulation.
• Previous work demonstrated their utility in vitro and in bacterial systems.

Purpose of the Study:

• To discover and engineer a bridge recombinase ortholog, ISCro4, for versatile human genome rearrangements.
• To optimize bridge systems for enhanced targeting specificity and efficiency in human cells.

Main Methods:

• Discovery and engineering of the ISCro4 bridge recombinase and its associated RNA.
• Rational engineering and deep mutational scanning of the recombinase and RNA components.
• Assays to measure DNA insertion efficiency and genome-wide specificity in human cells.
• Demonstration of intrachromosomal DNA inversion and excision.

Main Results:

• Engineered ISCro4 achieved up to 20% insertion efficiency in the human genome.
• Genome-wide specificity reached as high as 82% with optimized systems.
• Successfully demonstrated intrachromosomal inversion and excision of large DNA segments (up to 0.93 Mb).
• Proof-of-concept for excising disease-associated genetic elements.

Conclusions:

• ISCro4 is a powerful tool for programmable human genome engineering.
• Optimized bridge systems offer high efficiency and specificity for DNA rearrangements.
• Potential applications include therapeutic gene editing and removal of pathogenic genetic sequences.